The detection and identification of bacteria is of great interest in a variety of microbiological applications. For example the need to screen food, water and other beverages for pathogenic bacteria is crucial in ensuring consumer safety. The determination of levels of certain families of bacteria is a commonly used approach to estimating the shelf life and microbial acceptability of such products and hygienic status of the processing equipment and raw materials used in their manufacture. The diagnosis of microbial infections also relies on the detection of the causative organism. The screening of environmental waters for organisms such as Legionella has recently assumed considerable importance.
The desire to detect bacteriophages (viruses which specifically infect bacteria) stems from their ability to kill bacteria and hence the deleterious effect they can have on the fermentation of milk, for example, by killing the starter culture bacteria. Bacteriophages are also used, e.g. in the water industry, as tracers to determine the rate of river flow or sewage leakage.
The methods available to carry out bacterial detection and enumeration suffer from a number of drawbacks. Traditional culture based methods form the backbone of the tests used but as they rely on bacterial growth, often in selective media that allows the desired organism to grow while suppressing the growth of other bacteria, they are inherently slow; a total viable count taking 18-24 hours and detection of Salmonella taking 4-7 days. In many cases bacterial numbers may be under-estimated because their particular growth requirements may not be met by the media provided or they may have been sub-lethally injured or entered a stress induced physiological state in which they are viable but not culturable. Culture based methods are not suitable for on-site testing due to the long incubations required.
A variety of methods have been proposed to address these drawbacks and allow rapid bacterial detection, some claimed to be applicable to on-site use. For example, the measurement of adenosine triphosphate (ATP), an intracellular component of all living things, provides a rapid methodology but this is not specific and hence offers, at best, an estimation of the total bacterial population.
Immunoassay approaches with antibodies specific for the desired bacteria have failed to achieve widespread use because of inadequate specificity and sensitivity leading to the need for two days of enrichment culture before the immunoassay in the case of a Salmonella test, for example. Interference from competing organisms and the sample matrix have led to unacceptable rates of false positive and false negative results and protocols that are not substantially shorter than culture.
Methods based on DNA or RNA probes have been applied to bacterial detection but currently suffer from the problem of involving complicated protocols, unpleasant chemicals in some of the solutions and the need for elevated temperature. They certainly are not user friendly to technicians trained in classical microbiology. Together with immunoassays and nucleic acid amplification strategies such as the Polymerase Chain Reaction (PCR) they do not distinguish between live and dead bacteria. This makes them unsuitable for direct assays (where there is no culture step to allow the amplification of the living organisms) of the viable bacteria. In certain applications this distinction is very important e.g. when disinfectants have been used to ensure that there are very few living Legionella in a water system, it is meaningless to use an assay which fails to discriminate and detects the organisms which have been killed by the disinfection process.
There are a number of approaches that rely on expensive instruments to speed up the detection of bacteria. An example of these is impedance/conductance measurement where the bacterial presence is detected by their metabolism of complex nutrients to simpler chemicals with a concomitant change in the electrical properties of the medium. Such methods are highly capital intensive and inappropriate for small laboratories or on-site use.
Microscopy techniques, possibly employing selective staining, are limited in sensitivity and generally offer poor differentiation between living and dead bacteria. Routine microscopy will only permit presumptive identification based on morphology unless combined with selective culture or immunological staining.
In view of the above, it is highly desirable to have methods for the detection of bacteria and bacteriophages that are simple to perform, specific, rapid (providing results in hours rather than days), able to detect only living organisms, capable of on-site use and without the need for an expensive instrument. Preferably the assays would be performed on a wide variety of sample types without pre-treatment and with a minimum number of steps. The assay result should be a detectable event that is easily observed and amenable to automated reading. It would be further desirable if the assays were able to detect disabled bacteria which might otherwise require a pre-enrichment culture step and a selective enrichment step for detection. Non-culturable but viable organisms should also be detected.